Mitotic Spindle Microtubules Research Articles

Bistability and oscillations in cooperative microtubule and kinetochore dynamics in the mitotic spindle

In the mitotic spindle microtubules attach to kinetochores via catch bonds during metaphase, and microtubule depolymerization forces give rise to stochastic chromosome oscillations. We investigate the cooperative stochastic microtubule dynamics in spindle models consisting of ensembles of parallel microtubules, which attach to a kinetochore via elastic linkers. We include the dynamic instability of microtubules and forces on microtubules and kinetochores from elastic linkers. A one-sided model, where an external force acts on the kinetochore is solved analytically employing a mean-field approach based on Fokker–Planck equations. The solution establishes a bistable force–velocity relation of the microtubule ensemble in agreement with stochastic simulations. We derive constraints on linker stiffness and microtubule number for bistability. The bistable force–velocity relation of the one-sided spindle model gives rise to oscillations in the two-sided model, which can explain stochastic chromosome oscillations in metaphase (directional instability). We derive constraints on linker stiffness and microtubule number for metaphase chromosome oscillations. Including poleward microtubule flux into the model we can provide an explanation for the experimentally observed suppression of chromosome oscillations in cells with high poleward flux velocities. Chromosome oscillations persist in the presence of polar ejection forces, however, with a reduced amplitude and a phase shift between sister kinetochores. Moreover, polar ejection forces are necessary to align the chromosomes at the spindle equator and stabilize an alternating oscillation pattern of the two kinetochores. Finally, we modify the model such that microtubules can only exert tensile forces on the kinetochore resulting in a tug-of-war between the two microtubule ensembles. Then, induced microtubule catastrophes after reaching the kinetochore are necessary to stimulate oscillations. The model can reproduce experimental results for kinetochore oscillations in PtK1 cells quantitatively.

Mitotic Spindle Attachment to the Holocentric Chromosomes of Cuscuta europaea Does Not Correlate With the Distribution of CENH3 Chromatin.

The centromere is the region on a chromosome where the kinetochore assembles and spindle microtubules attach during mitosis and meiosis. In the vast majority of eukaryotes, the centromere position is determined epigenetically by the presence of the centromere-specific histone H3 variant CENH3. In species with monocentric chromosomes, CENH3 is confined to a single chromosomal region corresponding to the primary constriction on metaphase chromosomes. By contrast, in holocentrics, CENH3 (and thus centromere activity) is distributed along the entire chromosome length. Here, we report a unique pattern of CENH3 distribution in the holocentric plant Cuscuta europaea. This species expressed two major variants of CENH3, both of which were deposited into one to three discrete regions per chromosome, whereas the rest of the chromatin appeared to be devoid of CENH3. The two CENH3 variants fully co-localized, and their immunodetection signals overlapped with the positions of DAPI-positive heterochromatic bands containing the highly amplified satellite repeat CUS-TR24. This CENH3 distribution pattern contrasted with the distribution of the mitotic spindle microtubules, which attached at uniform density along the entire chromosome length. This distribution of spindle attachment sites proves the holocentric nature of C. europaea chromosomes and also suggests that, in this species, CENH3 either lost its function or acts in parallel to an additional CENH3-free mechanism of kinetochore positioning.

Clathrin's adaptor interaction sites are repurposed to stabilize microtubules during mitosis.

Clathrin ensures mitotic spindle stability and efficient chromosome alignment, independently of its vesicle trafficking function. Although clathrin localizes to the mitotic spindle and kinetochore fiber microtubule bundles, the mechanisms by which clathrin stabilizes microtubules are unclear. We show that clathrin adaptor interaction sites on clathrin heavy chain (CHC) are repurposed during mitosis to directly recruit the microtubule-stabilizing protein GTSE1 to the spindle. Structural analyses reveal that these sites interact directly with clathrin-box motifs on GTSE1. Disruption of this interaction releases GTSE1 from spindles, causing defects in chromosome alignment. Surprisingly, this disruption destabilizes astral microtubules, but not kinetochore-microtubule attachments, and chromosome alignment defects are due to a failure of chromosome congression independent of kinetochore-microtubule attachment stability. GTSE1 recruited to the spindle by clathrin stabilizes microtubules by inhibiting the microtubule depolymerase MCAK. This work uncovers a novel role of clathrin adaptor-type interactions to stabilize nonkinetochore fiber microtubules to support chromosome congression, defining for the first time a repurposing of this endocytic interaction mechanism during mitosis.

Structure of the Centromere Binding Factor 3 Complex from Kluyveromyces lactis

Kinetochores are the multiprotein complexes that link chromosomal centromeres to mitotic-spindle microtubules. Budding yeast centromeres comprise three sequential "centromere-determining elements", CDEI, II, and III. CDEI (8 bp) and CDEIII (∼25 bp) are conserved between Kluyveromyces lactis and Saccharomyces cerevisiae, but CDEII in the former is twice as long (160 bp) as CDEII in the latter (80 bp). The CBF3 complex recognizes CDEIII and is required for assembly of a centromeric nucleosome, which in turn recruits other kinetochore components. To understand differences in centromeric nucleosome assembly between K. lactis and S. cerevisiae, we determined the structure of a K. lactis CBF3 complex by electron cryomicroscopy at ∼4 Å resolution and compared it with published structures of S. cerevisiae CBF3. We show differences in the pose of Ndc10 and discuss potential models of the K. lactis centromeric nucleosome that account for the extended CDEII length.

Emerin Is Required for Proper Nucleus Reassembly after Mitosis: Implications for New Pathogenetic Mechanisms for Laminopathies Detected in EDMD1 Patients.

Emerin is an essential LEM (LAP2, Emerin, MAN1) domain protein in metazoans and an integral membrane protein associated with inner and outer nuclear membranes. Mutations in the human EMD gene coding for emerin result in the rare genetic disorder: Emery–Dreifuss muscular dystrophy type 1 (EDMD1). This disease belongs to a broader group called laminopathies—a heterogeneous group of rare genetic disorders affecting tissues of mesodermal origin. EDMD1 phenotype is characterized by progressive muscle wasting, contractures of the elbow and Achilles tendons, and cardiac conduction defects. Emerin is involved in many cellular and intranuclear processes through interactions with several partners: lamins; barrier-to-autointegration factor (BAF), β-catenin, actin, and tubulin. Our study demonstrates the presence of the emerin fraction which associates with mitotic spindle microtubules and centrosomes during mitosis and colocalizes during early mitosis with lamin A/C, BAF, and membranes at the mitotic spindle. Transfection studies with cells expressing EGFP-emerin protein demonstrate that the emerin fusion protein fraction also localizes to centrosomes and mitotic spindle microtubules during mitosis. Transient expression of emerin deletion mutants revealed that the resulting phenotypes vary and are mutant dependent. The most frequent phenotypes include aberrant nuclear shape, tubulin network mislocalization, aberrant mitosis, and mislocalization of centrosomes. Emerin deletion mutants demonstrated different chromatin binding capacities in an in vitro nuclear assembly assay and chromatin-binding properties correlated with the strength of phenotypic alteration in transfected cells. Aberrant tubulin staining and microtubule network phenotype appearance depended on the presence of the tubulin binding region in the expressed deletion mutants. We believe that the association with tubulin might help to “deliver” emerin and associated membranes to decondensing chromatin. Preliminary analyses of cells from Polish patients with EDMD1 revealed that for several mutations thought to be null for emerin protein, a truncated emerin protein was present. We infer that the EDMD1 phenotype may be strengthened by the toxicity of truncated emerin expressed in patients with certain nonsense mutations in EMD.

Architecture of the CBF3-centromere complex of the budding yeast kinetochore.

Kinetochores are multicomponent complexes responsible for coordinating the attachment of centromeric DNA to mitotic-spindle microtubules. The point centromeres of budding yeast are organized into three centromeric determining elements (CDEs), and are associated with the centromere-specific nucleosome Cse4. Deposition of Cse4 at CEN loci is dependent on the CBF3 complex that engages CDEIII to direct Cse4 nucleosomes to CDEII. To understand how CBF3 recognizes CDEIII and positions Cse4, we determined a cryo-EM structure of a CBF3−CEN complex. CBF3 interacts with CEN DNA as a head-to-head dimer that includes the whole of CDEIII and immediate 3’ regions. Specific CEN-binding of CBF3 is mediated by a Cep3 subunit of one of the CBF3 protomers that forms major groove interactions with the conserved and essential CCG and TGT motifs of CDEIII. We propose a model for a CBF3−Cse4−CEN complex with implications for understanding CBF3-directed deposition of the Cse4 nucleosome at CEN loci.

Chromokinesins

During the cell cycle it is critical that the duplicated DNA faithfully segregates to give rise to two genetically identical daughter cells. An even distribution of the genome during mitosis is mediated by mitotic spindle microtubules, assisted by, among others, motor proteins of the kinesin superfamily. Chromokinesins are members of the kinesin superfamily that harbour a specific DNA-binding domain. The best characterized chromokinesins belong to the kinesin-4/Kif4 and kinesin-10/Kif22 families, respectively. Functional analysis of chromokinesins in several model systems revealed their involvement in chromosome arm orientation and oscillations. This is consistent with their originally proposed role in the generation of polar ejection forces that assist chromosome congression to the spindle equator. Kinesin-12/Kif15 members comprise a third family of chromokinesins, but their role remains less understood. Noteworthy, all chromokinesins exhibit chromosome-independent localization on spindle microtubules, and recent works have significantly extended the portfolio of mitotic processes in which chromokinesins play a role, from error correction and DNA compaction, to the regulation of spindle microtubule dynamics.

LB1603 Centrosomes are required for the normal development of the skin epithelium

The skin epidermis has a stereotypical developmental and stratification program with specific cell division paradigms that may dictate progenitor cell fate. Centrosomes, the major microtubule organizing centers of animal cells, organize mitotic spindle microtubules and regulate cell division. During interphase and in differentiated cells, centrosomes provide the essential template for cilia. The functions of centrosomes during epidermal development have not been defined. In order to genetically determine and delineate the functions of mammalian centrosomes and cilia, we have conditionally removed Sas-4, which is essential for centrosome formation, or Ift88, which is essential for cilia but not centrosome formation, in the developing mouse skin epithelium. Our data showed that the skin epidermis without SAS-4 and centrosomes lacked cilia and was thinner with arrested hair follicle development at morphogenesis. As we have shown previously in the mouse embryo and developing brain, the loss of SAS-4/centrosomes led to a marked upregulation of p53 accompanied by cell death as early as embryonic day (E) 12.5; however, the differentiation process of the developing epidermis was not majorly affected. Interestingly, the removal of p53 in the SAS-4/centrosome-deficient epidermis was sufficient to rescue the epidermal and hair follicle defects but not the loss of cilia. The phenotype observed upon centrosome removal was also not due to the secondary loss of cilia because an IFT88/cilia epidermal knockout did not show overt developmental phenotypes. We conclude that in contrast to cilia, centrosomes in the epidermis are essential to ensure proper epidermal and hair follicle development in a p53-dependent manner.

Cdt1 stabilizes kinetochore-microtubule attachments via an Aurora B kinase-dependent mechanism.

Robust kinetochore-microtubule (kMT) attachment is critical for accurate chromosome segregation. G2/M-specific depletion of human Cdt1 that localizes to kinetochores in an Ndc80 complex-dependent manner leads to abnormal kMT attachments and mitotic arrest. This indicates an independent mitotic role for Cdt1 in addition to its prototypic function in DNA replication origin licensing. Here, we show that Cdt1 directly binds to microtubules (MTs). Endogenous or transiently expressed Cdt1 localizes to both mitotic spindle MTs and kinetochores. Deletion mapping of Cdt1 revealed that the regions comprising the middle and C-terminal winged-helix domains but lacking the N-terminal unstructured region were required for efficient MT binding. Mitotic kinase Aurora B interacts with and phosphorylates Cdt1. Aurora B-phosphomimetic Cdt1 exhibited attenuated MT binding, and its cellular expression induced defective kMT attachments with a concomitant delay in mitotic progression. Thus we provide mechanistic insight into how Cdt1 affects overall kMT stability in an Aurora B kinase phosphorylation-dependent manner; which is envisioned to augment the MT-binding of the Ndc80 complex.

Direct interactions of mitotic arrest deficient 1 (MAD1) domains with each other and MAD2 conformers are required for mitotic checkpoint signaling

As a sensitive signaling system, the mitotic checkpoint ensures faithful chromosome segregation by delaying anaphase onset even when a single kinetochore is unattached to mitotic spindle microtubules. The key signal amplification reaction for the checkpoint is the conformational conversion of "open" mitotic arrest deficient 2 (O-MAD2) into "closed" MAD2 (C-MAD2). The reaction has been suggested to be catalyzed by an unusual catalyst, a MAD1:C-MAD2 tetramer, but how the catalysis is executed and regulated remains elusive. Here, we report that in addition to the well-characterized middle region of MAD1 containing the MAD2-interaction motif (MIM), both N- and C-terminal domains (NTD and CTD) of MAD1 also contribute to mitotic checkpoint signaling. Unlike the MIM, which stably associated only with C-MAD2, the NTD and CTD in MAD1 surprisingly bound both O- and C-MAD2, suggesting that these two domains interact with both substrates and products of the O-to-C conversion. MAD1NTD and MAD1CTD also interacted with each other and with the MPS1 protein kinase, which phosphorylated both NTD and CTD. This phosphorylation decreased the NTD:CTD interaction and also CTD's interaction with MPS1. Of note, mutating the phosphorylation sites in the MAD1CTD, including Thr-716, compromised MAD2 binding and the checkpoint responses. We further noted that Ser-610 and Tyr-634 also contribute to the mitotic checkpoint signaling. Our results have uncovered that the MAD1NTD and MAD1CTD directly interact with each other and with MAD2 conformers and are regulated by MPS1 kinase, providing critical insights into mitotic checkpoint signaling.

Microtubule Sliding within the Bridging Fiber Pushes Kinetochore Fibers Apart to Segregate Chromosomes

SummaryDuring cell division, mitotic spindle microtubules segregate chromosomes by exerting forces on kinetochores. What forces drive chromosome segregation in anaphase remains a central question. The current model for anaphase in human cells includes shortening of kinetochore fibers and separation of spindle poles. Both processes require kinetochores to be linked with the poles. Here we show, by combining laser ablation, photoactivation, and theoretical modeling, that kinetochores can separate without any attachment to one spindle pole. This separation requires the bridging fiber, a microtubule bundle that connects sister kinetochore fibers. Bridging fiber microtubules in intact spindles slide apart with kinetochore fibers, indicating strong crosslinks between them. We conclude that sliding of microtubules within the bridging fibers drives pole separation and pushes kinetochore fibers poleward by the friction of passive crosslinks between these fibers. Thus, sliding within the bridging fiber works together with the shortening of kinetochore fibers to segregate chromosomes.

Identification of pyrrolopyrimidine derivative PP-13 as a novel microtubule-destabilizing agent with promising anticancer properties

Despite the emergence of targeted therapies and immunotherapy, chemotherapy remains the gold-standard for the treatment of most patients with solid malignancies. Spindle poisons that interfere with microtubule dynamics are commonly used in chemotherapy drug combinations. However, their troublesome side effects and the emergence of chemoresistance highlight the need for identifying alternative agents. We performed a high throughput cell-based screening and selected a pyrrolopyrimidine molecule (named PP-13). In the present study, we evaluated its anticancer properties in vitro and in vivo. We showed that PP-13 exerted cytotoxic effects on various cancer cells, including those resistant to current targeted therapies and chemotherapies. PP-13 induced a transient mitotic blockade by interfering with both mitotic spindle organization and microtubule dynamics and finally led to mitotic slippage, aneuploidy and direct apoptotic death. PP-13 was identified as a microtubule-targeting agent that binds directly to the colchicine site in β-tubulin. Interestingly, PP-13 overcame the multidrug-resistant cancer cell phenotype and significantly reduced tumour growth and metastatic invasiveness without any noticeable toxicity for the chicken embryo in vivo. Overall, PP-13 appears to be a novel synthetic microtubule inhibitor with interesting anticancer properties and could be further investigated as a potent alternative for the management of malignancies including chemoresistant ones.

Kinetochore Components Required for Centromeric Chromatin Assembly Are Impacted by Msc1 in Schizosaccharomyces pombe.

Eukaryotic chromosome segregation requires a protein complex known as the kinetochore that mediates attachment between mitotic spindle microtubules and centromere-specific nucleosomes composed of the widely conserved histone variant CENP-A. Mutations in kinetochore proteins of the fission yeast Schizosaccharomyces pombe lead to chromosome missegregation such that daughter cells emerge from mitosis with unequal DNA content. We find that multiple copies of Msc1-a fission yeast homolog of the KDM5 family of proteins-suppresses the temperature-sensitive growth defect of several kinetochore mutants, including mis16 and mis18, as well as mis6, mis15, and mis17, components of the Constitutive Centromere Associated Network (CCAN). On the other hand, deletion of msc1 exacerbates both the growth defect and chromosome missegregation phenotype of each of these mutants. The C-terminal PHD domains of Msc1, previously shown to associate with a histone deacetylase activity, are necessary for Msc1 function when kinetochore mutants are compromised. We also demonstrate that, in the absence of Msc1, the frequency of localization to the kinetochore of Mis16 and Mis15 is altered from wild-type cells. As we show here for msc1, others have shown that elevating cnp1 levels acts similarly to promote survival of the CCAN mutants. The rescue of mis15 and mis17 by cnp1 is, however, independent of msc1 Thus, Msc1 appears to contribute to the chromatin environment at the centromere: the absence of Msc1 sensitizes cells to perturbations in kinetochore function, while elevating Msc1 overcomes loss of function of critical components of the kinetochore and centromere.

GTSE1 regulates spindle microtubule dynamics to control Aurora B kinase and Kif4A chromokinesin on chromosome arms.

In mitosis, the dynamic assembly and disassembly of microtubules are critical for normal chromosome movement and segregation. Microtubule turnover varies among different mitotic spindle microtubules, dictated by their spatial distribution within the spindle. How turnover among the various classes of spindle microtubules is differentially regulated and the resulting significance of differential turnover for chromosome movement remains a mystery. As a new tactic, we used global microarray meta-analysis (GAMMA), a bioinformatic method, to identify novel regulators of mitosis, and in this study, we describe G2- and S phase-expressed protein 1 (GTSE1). GTSE1 is expressed exclusively in late G2 and M phase. From nuclear envelope breakdown until anaphase onset, GTSE1 binds preferentially to the most stable mitotic spindle microtubules and promotes their turnover. Cells depleted of GTSE1 show defects in chromosome alignment at the metaphase plate and in spindle pole integrity. These defects are coupled with an increase in the proportion of stable mitotic spindle microtubules. A consequence of this reduced microtubule turnover is diminished recruitment and activity of Aurora B kinase on chromosome arms. This decrease in Aurora B results in diminished binding of the chromokinesin Kif4A to chromosome arms.

Theoretical model of mitotic spindle microtubule growth for FRAP curve interpretation

BackgroundSpindle FRAP curves depend on the kinetic parameters of microtubule polymerization and depolymerization. The empirical FRAP curve proposed earlier permits determination of only one such dynamic parameter, commonly called the "tubulin turnover". The aim of our study was to build a FRAP curve based on an already known kinetic model of microtubule growth.ResultsA numerical expression that describes the distribution of polymerizing and depolymerizing microtubule ends as a function of four kinetic parameters is presented. In addition, a theoretical FRAP curve for the metaphase spindle is constructed using previously published dynamic parameters.ConclusionThe numerical expression we elaborated can replace the empirical FRAP curve described earlier for a spindle comprising fluorescently marked microtubules. The curve we generated fits well the experimental data.

115 Centrosomes are required for the development of the skin epidermis and hair folllicles

Centrosomes are the major microtubule organizing centers of animal cells and are composed of a pair of centrioles that are surrounded by a pericentriolar matrix of proteins. Centrosomes organize mitotic spindle microtubules and provide the essential centriolar templates for cilia and flagella. Mutations in genes encoding centrosomal proteins in humans cause syndromes characterised by primordial dwarfism and microcephaly. In order to genetically define the functions of mammalian and centrosomes and model the human diesease, we have recently conditionally removed Sas-4, which is essential for centrosome formation, in the developing mouse brain. Our data showed that the neural progenitors without centrosomes activated p53-dependent cell death; but also detached from their normal niche at the ventricle and randomized their mitotic spindle orientation; however cortical architecture was largely preserved. The functions of mammalian centrosomes in other developing tissues and in stem cells are not known. The skin epidermis and hair follicles have well-defined developmental and differentiation programs with specific cell division paradigms and stem cell populations. Therefore, we used the developing skin epidermis and hair follicles to dissect the functions of centrosomes in stem cell division and differentiation. Our data showed that the skin epidermis without SAS-4 and centrosomes lacked cilia and showed epithelial defects and hair loss. The mutant epidermis without centrosomes was thinner, showed aberrant differentiation, and arrested hair follicle development at morphogenesis. These phenotypes were not due to the secondary loss of cilia upon centrosome removal because a cilia-specific epidermal knockout did not show any overt phenotypes. Interestingly, The removal of only one copy of p53 in the centrosome-deficient skin was sufficient to significantly rescue the epidermal and hair follicle defects but not the loss of cilia. We conclude that in contrast to cilia, centrosomes in the epidermis are essential to ensure proper epidermal and hair follicle development in a p53-dependent manner.

Nup62, associated with spindle microtubule rather than spindle matrix, is involved in chromosome alignment and spindle assembly during mitosis.

An increasing number of the active mitotic functions of nucleoporins in the distinct steps of mitosis have been assigned over the past few years. As one of FG-repeats containing nucleoporins, Nup62 has been found to be involved in nuclear transport, cell migration, virus infection, and cell cycle regulation. However, the role and mechanism of Nup62 in mitotic regulation have not been fully revealed. In this paper, it was revealed that a fraction of Nup62 was associated with mitotic spindle microtubule instead of spindle matrix, and the localization of Nup62 in the mitotic spindle depended on its three coiled-coil domains rather than Crm1, although Nup62 strongly interacted with Crm1 during mitosis. Moreover, depletion of Nup62 by small interference of RNA seriously induced the defects of chromosome alignment and spindle assembly although the bipolar spindle was observed in most of the Nup62 knock-down cells. Notably, congression of polar chromosome defect was observed in more than 30% of Nup62 knock-down cells. These findings revealed that Nup62 was a novel mitotic spindle associated nucleoporin and involved in chromosome alignment and spindle assembly.

Allosteric modulation of AURKA kinase activity by a small-molecule inhibitor of its protein-protein interaction with TPX2.

The essential mitotic kinase Aurora A (AURKA) is controlled during cell cycle progression via two distinct mechanisms. Following activation loop autophosphorylation early in mitosis when it localizes to centrosomes, AURKA is allosterically activated on the mitotic spindle via binding to the microtubule-associated protein, TPX2. Here, we report the discovery of AurkinA, a novel chemical inhibitor of the AURKA-TPX2 interaction, which acts via an unexpected structural mechanism to inhibit AURKA activity and mitotic localization. In crystal structures, AurkinA binds to a hydrophobic pocket (the ‘Y pocket’) that normally accommodates a conserved Tyr-Ser-Tyr motif from TPX2, blocking the AURKA-TPX2 interaction. AurkinA binding to the Y- pocket induces structural changes in AURKA that inhibit catalytic activity in vitro and in cells, without affecting ATP binding to the active site, defining a novel mechanism of allosteric inhibition. Consistent with this mechanism, cells exposed to AurkinA mislocalise AURKA from mitotic spindle microtubules. Thus, our findings provide fresh insight into the catalytic mechanism of AURKA, and identify a key structural feature as the target for a new class of dual-mode AURKA inhibitors, with implications for the chemical biology and selective therapeutic targeting of structurally related kinases.

Synergistic role of fission yeast Alp16GCP6 and Mzt1MOZART1 in γ-tubulin complex recruitment to mitotic spindle pole bodies and spindle assembly.

In fission yeast, γ-tubulin ring complex (γTuRC)-specific components Gfh1(GCP4), Mod21(GCP5), and Alp16(GCP6) are nonessential for cell growth. Of these deletion mutants, only alp16Δ shows synthetic lethality with temperature-sensitive mutants of Mzt1(MOZART1), a component of the γTuRC required for recruitment of the complex to microtubule-organizing centers. γ-Tubulin small complex levels at mitotic spindle pole bodies (SPBs, the centrosome equivalent in fungi) and microtubule levels for preanaphase spindles are significantly reduced in alp16Δ cells but not in gfh1Δ or mod21Δ cells. Furthermore, alp16Δ cells often form monopolar spindles and frequently lose a minichromosome when the spindle assembly checkpoint is inactivated. Alp16(GCP6) promotes Mzt1-dependent γTuRC recruitment to mitotic SPBs and enhances spindle microtubule assembly in a manner dependent on its expression levels. Gfh1(GCP4) and Mod21(GCP5) are not required for Alp16(GCP6)-dependent γTuRC recruitment. Mzt1 has an additional role in the activation of the γTuRC for spindle microtubule assembly. The ratio of Mzt1 to γTuRC levels for preanaphase spindles is higher than at other stages of the cell cycle. Mzt1 overproduction enhances spindle microtubule assembly without affecting γTuRC levels at mitotic SPBs. We propose that Alp16(GCP6) and Mzt1 act synergistically for efficient bipolar spindle assembly to ensure faithful chromosome segregation.

An ensemble of specifically targeted proteins stabilizes cortical microtubules in the human parasite Toxoplasma gondii.

Although all microtubules within a single cell are polymerized from virtually identical subunits, different microtubule populations carry out specialized and diverse functions, including directional transport, force generation, and cellular morphogenesis. Functional differentiation requires specific targeting of associated proteins to subsets or even subregions of these polymers. The cytoskeleton of Toxoplasma gondii, an important human parasite, contains at least five distinct tubulin-based structures. In this work, we define the differential localization of proteins along the cortical microtubules of T. gondii, established during daughter biogenesis and regulated by protein expression and exchange. These proteins distinguish cortical from mitotic spindle microtubules, even though the assembly of these subsets is contemporaneous during cell division. Finally, proteins associated with cortical microtubules collectively protect the stability of the polymers with a remarkable degree of functional redundancy.